Esters of alpha-aminobenzylpenicillin in dosage unit form

ABSTRACT

THE INVENTION RELATES TO A PHARMACEUTICAL COMPOSITION HAVING ANTIBACTERIAL ACTIVITY, AND TO THE COMPOSITION IN DOSAGE UNIT FORM FOR USE IN THE TREATMENT OF INFECTIOUS DESEASES, COMPRISING, AS AN ACTIVE INGREDIENT A NEW ESTER OF A-AMINOBENZYLPENICILLIN OF THE GENERAL FORMULA:   2-(A-(CH2)N-COO-CH2-OOC-),3,3-DI(CH3-),6-(PHENYL-CH(-NH2)-   CO-NH-)PENAM   IN WHICH N IS AN INTEGER FROM 0 TO 5, AND A IS AN UNSUBSTITUTED OR SUBSTITUTED ALIPHATIC, ALICYCLIC, AROMATIC, OR HETEROCYCYLIC RADICAL, OR A SALT OF SUCH ESTER WITH A PHARMACEUTICALLY ACCEPTABLE ACID, AND AN ATOXIC, PHARMACEUTICALLY ACCEPTABLE CARRIER, THE QUANTITY OF THE ACTIVE INGREDIENT IN A DOSE BEING BETWEEN 100 MG. AND 800 MG. THE INVENTION ALSO RELATES TO A METHOD OF TREATING PATIENTS SUFFERING FROM INFECTIOUS DISEASES WITH THE DOSAGE UNITS IN QUESTION, ADMINISTERING FROM 500 TO 5000 MG. PER DAY OF THE ACTIVE COMPONENT.

United States Patent O 3,660,575 ESTERS OF a-AMINOBENZYLPENICILLIN IN DOSAGE UNIT FORM Erling Knud Frederiksen, Holte, and Wagn Ole Godtfredsen, Vaerlose, Denmark, assignors to Lovens kemiske Fabrik Produktionsaktieselskab, Ballerup, Denmark No Drawing. Filed Sept. 26, 1968, Ser. No. 763,010 Claims priority, application Great Britain, Sept. 29, 1967, 44,535/67; Oct. 5, 1967, 45,600/67; Oct. 23, 1967, 48,127/67; Nov. 10, 1967, 51,358/67; Dec. 6, 1967, 55,489/67; Jan. 3, 1968, 499/68; Mar. 22, 1968, 14,041/68; Mar. 29, 1968, 15,312/68 Int. Cl. A61k 21/00 U.S. Cl. 424-271 4 Claims ABSTRACT OF THE DISCLOSURE The invention relates to a pharmaceutical composition having antibacterial activity, and to the composition in dosage unit form for use in the treatment of infectious diseases, comprising, as an active ingredient a new ester of a-aminobenzylpenicillin of the general formula:

This invention relates to a pharmaceutical composition having antibacterial activity, and to the composition in dosage unit form, containing at least one member of a group of new esters of a-aminobenzylpenicillin, these esters having the formula s QoHcONHZH-CH 0133 in which n is an integer from 0 to 5; and A is an unsubstituted or substituted aliphatic, alicyclic, aromatic, or heterocyclic radical, to salts of these esters with pharmaceutically acceptable acids, and to methods of preparing these new esters.

More particularly, A may represent an aliphatic hydrocarbon radical in which the carbon chain can be straight or branched, saturated or unsaturated, having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec. and tert. butyl, pentyl, hexyl, and the like; an alicyclic, carboxylic radical having from 3 to 10 carbon atoms as ring members, and in which the ring or rings may be saturated or may contain one or two double bonds, depending on the number of carbon atoms, such as cycloice pentyl, cyclohexyl, l-adamantyl, 1-bicyclo(2.2.2)octyl, cyclopentenyl, cyclohexenyl, in which the double bond may be placed in the 2,3- or 3,4-position, and the like; an aromatic radical such as a monocyclic carbocyclic aryl radical, e.g. a phenyl or substituted phenyl radical, a bicyclic carbocyclic aryl radical, e.g. lor Z-naphthyl or substituted naphthyl radical; a heterocyclic aryl radical, which may contain from 5 to 10 atoms as ring members, such as pyridyl, pyrazinyl, pyrimidyl, thienyl, furyl, or quinolyl, in which the hetero atom may be in any of the available positions, and which may further have substituents in one or more of the remaining positions. As already mentioned, the radical A may have further substituents, such as lower alkyl, e.g. methyl, ethyl, propyl, isopropyl, butyl and the like; lower alkoxy, e.g. methoxy, ethoxy and the like; lower alkylthio, e.g. methylmercapto or ethylmercapto; halo-lower alkyl, e.g. mono, dior trifluoromethyl, mono-, dior trichloromethyl or the ethyl homologues and the corresponding bromo derivatives; halogens, e.g. fluorine, bromine or chlorine; or nitro groups. The substituents may be placed in any possible positions. Salts of the new esters may be formed with inorganic acids, e.g. hydrochloric, hydrobromic, hydroiodic acid and sulphuric acid and the like; and organic acids such as citric, tartaric, maleic acid and the like.

The said esters and their salts as well as methods of preparing them have been described and claimed in our concurrently filed patent application Ser. No. 762,987, the disclosure of which is included herein by reference to the said specification.

The acid-resistant a-aminobenzylpenicillin is a wellknown and widely used broad-spectrum antibiotic, which on oral administration, however, is insufficiently absorbed in the organism.

Acyloxy methyl esters of other penicillins are also known, but are practically not absorbed in the organism on oral administration, except for the acetoxymethyl ester of benzylpenicillin, which is absorbed to a certain degree, giving rise to low, but prolonged blood concentrations.

It is a surprising and important feature of the present compositions that the therapeutically active ingredient is absorbed much faster and more efiiciently than a-aminobenzylpenicillin, and is hydrolyzed rapidly into the latter compound by deacylases present in the body fluids, and by deacylases produced by micro-organisms, e.g. pathogenic micro-organisms, giving rise to much higher antibacterial blood and tissue levels than does the administration of a corresponding amount of a-aminobenzylpenicillin. The improved absorption is also reflected in an increased excretion of a-aminobenzylpenicillin in the urine.

The improved resorption is evidenced, in particular, in a preferred active ingredient of the compositions of the invention, pivaloyloxymethyl D(-)-a-aminobenzylpenicillinate.

In the following Tables I and II the resorption of the ester in question has been more elaborately illustrated and compared with D -a-aminobenzylpenicillin.

TABLE I Serum concentrations of D()-u-aminobenzylpenicillin g/ml.) after oral administration of D(--)-a-aminobenzylpencillin (2.50 mg.) (column A) and pivaloyloxymethyl D,( )-a-aminobenzylpenicillinate (350 mg.) (column B) to healthy volunteers.

% hour 1 hour 2 hours 4 hours 6 hours B A B A B A B A B TABLE II Absorption and distribution in blood and organs from rats after oral administration of (A) D(-)-u-aminobenzylpenicillin (100 mg./kg.) (B) Pivaloyloxymethyl D(-)-a-aminobenzylpenicillinate,

HCl (143 mg./kg.)

g. per ml. or per g. wet weight of tissue 5 hour 1 hour 2 hours A B A B A B Thus, it is an object of the present invention to provide an antibacterial composition for use in the treatment of infectious diseases, which gives rise to a comparatively high level of a-aminobenzylpenicillin in the blood and various organs after oral or parenteral administration, which contains as an active ingredient, an ester of a-aminobenzylpenicillin of the Formula I given hereinbefore, or a salt of such ester with an atoxic pharmaceutically acceptable inorganic or organic acid, mixed up with a solid carrier and/or auxiliary agent.

In the said composition, the proportion of therapeutically active material to carrier substance and auxiliary agent can vary between 1% and 95%. The composition in question can either be worked up to pharmaceutical forms of presentation such as tablets, pills or drages, or the composition can be filled in medical containers such as capsules. Pharmaceutical organic or inorganic, solid or liquid carriers suitable for enteral or parenteral adminstration can be used to make up the composition. Gelatine, lactose, starch, magnesium stearate, talc, vegetable and animal fats, gum, polyalkylene glycol, or other known carriers for medicaments are all suitable as carriers. The preferred salt of the ester of Formula I is the hydrochloride but other inorganic or organic salts may be used, e.g. the phosphate, the acetate and the like. Furthermore the composition may contain other pharmaceu tically active components which can appropriately be administered together with the ester or salt in the treatment of infectious diseases, such as other suitable antibiotics.

Another object of the invention resides in the selection of a dose of the said ester or salt which can be administered so that the desired activity is achieved without simultaneous secondary effects.

It has been found that the esters of Formula I or their salts are conveniently administered in dosage units containing not less than 0.025 gm. and not more than -1.0 gm., preferably from 100 mg. to 800 mg. in total of ester. By the term dosage unit is meant a unitary, i.e. a single does capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically stable unit dose, comprising either the active material as such or a mixture of it with a solid pharmaceutical carrier.

In the form of such dosage unit the compound may be administered one or more times a day at appropriate intervals, always depending, however, on the condition of the patient.

As a non-limiting example of a dosage unit, the pre scription below describes the preparation of tablets each containing 0.360 gm. of pivaloyloxymethyl D()-u aminobenzylpenicillinate hydrochloride.

Ingredients: Gm. Pivaloyloxymethyl D()-oz aminobenzylpenicillinate hydrochloride 360 Lactose Polyvinylpyrrolidone 7 Corn starch 50 Talc 25 Magnesium stearate 3 The pivaloyloxymethyl ester and the lactone are screened through a 20 mesh per linear inch sieve and mixed together for 15 minutes. Thereafter the mixed powders are Wetted with a solution of polyvinylpyrrolidone in 96% ethanol. The moist mass is passed through a 10 mesh per linear inch screen and then dried at 38 degrees centigrade. When the alcohol has evaporated, the granules are broken on a 16 mesh per linear inch sieve and mixed with the corn starch, talc and magnesium stearate. The granulate is compressed into tablets of 0.50 gram Weight using inch punches and dies, yielding 1000 tablets each containing 0.360 gram of the pivaloyloxymethyl ester.

In a particularly preferred form of administration, capsules are employed of, for instance gelatine or another easily digestible or disintegrable material containing from 0.1 to 1.0 gram of the ester, e.g. the pivaloyloxymethyl ester, if necessary mixed with a minor amount of auxiliary substances in order to obtain a free-flowing powder suitable for filling into the capsules. Such capsules are conveniently prepared in accordance with the following description.

Ingredients: Gm. Pivaloyloxymethyl D -u-amino benzylpenicillinate hydrochloride 360 Magnesium stearate 3 Talc 17 The ingredients are passed through a mesh per linear inch sieve and mixed for 15 minutes. The mixture is filled into No. 0 gelatine capsules (Parke, Davis & (10.), using a semi-automatic capsule-filling machine shaken by vibrator. Each capsule contains 380 mg. of the mixture corresponding to 360 mg. of the pivaloyloxymethyl ester.

The invention further comprises a method of treating patients suffering from infectious diseases, which comprises daily administration of a number of the dosage units of this invention to each patient corresponding to from 500 mg. to 5000 mg. of the therapeutically active ingredient.

The present invention also includes methods for the preparation of the new esters and their salts. Owing to the presence of the a-amino group of the side chain and the hydrolyzable ester group of the compounds of the invention, certain considerations have to be taken in the preparation.

One method comprises a first step in which an a-substituted benzylpenicillin derivative of the Formula II is reacted with a compound of Formula III according to the following scheme:

6 may be free or substituted, for instance with a trimethylsilyl radical. Some of the starting substances of Formula In these formulae, n and A are as defined hereinbefore; R is an amino group, or a substituted amino group ZNH, or a group in which can be converted into an amino group, such as an azido or nitro group, or a halogen atom; COOY and XCH represent radicals capable of reacting with each other to form the COOCH grouping. As examples of such radicals, Y may be hydrogen or an alkali metal or a tertiary ammonium group and X may be a halogen atom, preferably chlorine or bromine, an acyloxy radical having from 1 to 16 carbon atoms, or an alkylsulphonyloxy or arylsulphonyloxy radical. The compounds formed by the above-mentioned reaction are esters of a-R-substituted benzylpenicillins (IV). When R is NH Formula IV represents the compounds of the invention, whereas when R has the other meanings defined above, Formula IV represents interesting intermediates in the synthesis of the compound of the invention, and as such constitute a part of this invention. As a common characteristic of the substituent R, it can be said that it is selected from groups which after the above reaction are capable of being converted into an amino group by means of methods mild enough to avoid a cleavage of the molecule at the ester group or at the lactam ring. In particular, the substituent R may have the formula Z--NH- where Z is a benzoyloxycarbonyl radical, a p-halo-, p-nitro-, or p-methoxybenzyloxycarbonyl radical, a 8, S,B-trichloroethyloxycarbonyl or an allyloxycarbonyl radical; or Z is a sulphurcontaining radical, such as tritylsulphenyl radical, or an arylsulphenyl radical, e.g. an o-nitropheuylsulphenyl radical; Z may also be a triphenylmethyl (also called trityl) radical, a tertiary butoxycarbonyl radical, or a radical obtained by reacting the free amino group with a ,B-dicarbonyl compound such as acetylacetone, acetoacetic esters or benzoyl-acetone to form enamines or Schiif bases. In general, any group represented by Z, which can be split off by reduction, by mild acid hydrolysis or by other mild reactions known per se will be suitable, since experiments have shown, that the esters of Formula I formed by the reaction in question are stable under such conditions. As examples of R substituents which can be converted into an amino group mention can be made of the azido group, the nitro group and halogen atoms, for instance a bromine atom. The starting compounds of Formula II, in which R is different from NH are known as intermediates in the synthesis of a-aminobenzylpenicillin. They exist in two epimeric forms. If the starting materials are prepared in form of the D or the L epimers, the corresponding epimeric form of the compounds of the invention will be obtained. If, on the other hand, a mixture of the epimeric forms of the starting compound is used, a mixture is obtained. This mixture can be separated in the two epimers, for instance, by fractional crystallization.

The methods of preparing the starting substances of Formula II are standard procedures employed in peptide chemistry and include, for instance, the conversion of the phenylacetic acid to the R-substituted phenylacetic acid, R having the meaning given before, followed by a reaction between a reactive derivative of this intermediate and 6-aminopenicillanic acid in which the amino group II can also be prepared from ot-aminobenzylpenicillin or salts thereof.

Some of the starting compounds of Formula III are known compounds, the preparation of which is described in the literature. Others are new, but can be prepared in the same way as the known, using methods which are standard procedures for this type of compounds.

Among such methods may be mentioned the reaction of an acid halide with paraformaldehyde (as described in e.g. J.A.C.S. 43, 660 (1921)) or the halogenation of methyl esters (as described in e.g. Acta Chem. Scand. 20, 1273 (1966) and references cited there).

The reaction of the compouunds of Formula II with the compounds of Formula III can be performed at or below room temperature or by gentle heating up to the boiling point of the solvent, depending on the meaning of Y and X. Different organic solvents or mixtures thereof with water may be used, for example, acetone, dioxane, tetrahydrofuran, methylene chloride and dimethylformamide. The reaction products are crystalline or oily products, which can be used in the next step Without further purification. By repeated reprecipitations, the oily products can be converted into crystalline or amorphous powders.

The subsequent reaction step (IV-I), by which the group R is converted into an amino group, may be effected by different methods known from peptide synthesis, depending on what R stands for.

Catalytic hydrogenation will be preferred when R has the formula ZNH, and Z stands for benzyloxycarbonyl and related derivatives thereof, and for trityl. This hydrogenation is preferably performed at room temperature and at atmospheric or slightly elevated pressure in a solvent which may be a non-reducible organic solvent, or a mixture thereof with Water. The preferred catalysts are noble metal catalysts, such as palladium or platinum, or Raney nickel, but other catalysts can be used as well. Electrolytic reduction can also be used in these cases, When Z stands for a fi,fl,fi-trichloroethyloxycarbonyl group, a reduction with Zn in acetic acid is preferable. A mild acid hydrolysis is preferred in the case where Z stands for a sulphur-containing radical, an enamine or a Schiff base, for instance by means of a dilute solution of hydrogen chloride in aqueous acetone at a pH of about 2. A treatment with formic acid at room temperature is especially suitable for the removal of Z, when Z is a tertiary butoxycarbonyl radical. Also known from the literature is the removal of the o-nitriphenylsulphenyl radical involving a nucleophilic attack on the sulphur atom of the sulphenamide group, the best yield in the present case being obtained with sodium or potassium iodide, sodium thiosulphate, sodium hydrogen sulphide, sodium dithionite, or potassiumthiocyanate. Other sulphenamide radicals can be split off in the same Way. If R is an azido or nitro group, or a halogen atom, especially a bromine atom, these may be transformed into the free amino group in known manner, the azido and the nitro group by a catalytic hydrogenation with a noble metal catalyst, or with Raney nickel, or by an electrolytic reduction, and the halogen atom by an amination, for instance with hexamethylenetetramine.

Another suitable method for the preparation of the compounds of the present invention comprises the reaction of a reactive derivative of an tat-substituted phenylacetic acid of Formula V with an ester of 6-aminopenicillanic acid of Formula VI whereby a product of the above Formula IV is obtained according to the following scheme:

In Formula V, R has the same meaning as in Formula II; in Formula VI, 11 and A have the same meaning as defined in Formula I; and the radicals CO-Y and X -HN represent radicals capable of reacting with each other to form a CONH-bridge. For instance COY can be the radical of an acid halide, such as an acid chloride or bromide; an anhydride; a mixed anhydride with an alkyl carbonic acid, such as isobutyl-carbonic acid; a carboxylic acid; an inorganic acid or a sulphonic acid; or a radical obtained by reacting the a-substituted phenylacetic acid with a carbodiimide, or N,N'-carbonyldiimidazole, or a similarly functioning compound; X can be hydrogen, or a trialkylsilyl group, the alkyl having not more than carbon atoms.

The reaction can be performed in an organic solvent or in a mixture thereof with water either at a low temperature or at slightly elevated temperature. Suitable solvents are methylene chloride, chloroform, ethyl acetate, acetone, dimethylformamide, or dimethylacetamide, ether, tetrahydrofuran, dioxane, or similar inert solvents.

The reaction products are isolated in conventional manner, e.g. by re-precipitation or by removal of the solvent followed by recrystallization from a solvent. The starting compounds of Formula V are known compounds which can be prepared by standard methods known from the peptide chemistry.

The compounds of Formula VI are new compounds, which are interesting intermediates in the synthesis of the compounds of Formula I and as such constitute a part of this invention. They can be prepared by the treatment of 6-amino-penicillanic acid in the form of a salt, such as an alkali metal salt or the triethylammonium salt, with a halomethyl ester of the formula R CH OCO(CH ),,-A (VII) in which R is a halogen, preferably a chlorine or bromine atom, or a sulphonyloxy radical, such as methane sulphonyloxy or toluenesulphonyloxy radical, and n and A are defined above. The 6-amino-penicillanic acid may be used as such, or the 6-amino group may be protected during the esterification process. Only protecting radicals, which can be easily removed without causing any cleavage of the lactam ring or the ester group, are suitable in this case, for instance triphenylmethyl or trimethylsilyl radicals. The reaction is performed in an inert organic solvent such as acetone, dimethylformamide, or methylene chloride, and at or below room temperature, or at slightly elevated temperatures. When the amino group has been protected, the removal of the protecting group can be performed by different methods, such as hydrogenolysis or hydrolysis under neutral or acidic conditions which do not attack the ,B-lactam ring and the ester group. The reaction products of Formula VI (X=H) are conveniently isolated as their acid addition salts with, for instance, p-toluenesulphonic acid or other inorganic or organic acids such as sulphuric, phosphoric, hydrocholric, acetic, maleic, tartaric and the like acids.

In another embodiment of the process, the compounds of Formula VI can be prepared by esterification of any of the industrially accessible penicillins, or preferably their salts, with a compound of the aforementioned Formula VII under similar conditions as already described, whereafter the side chain of the resulting penicillin ester is split off to yield the 6-amino-penicillanic ester of Formula VI, or a salt thereof.

The cleavage of the amide bond can be performed by a modification of the procedure described in Belgian patent specification No. 698,596 by reacting the 6-acylaminopenicillanic acid ester with an acid halide in the presence of an acid-binding agent, such as quinoline or pyridine, and the like. The preferred halide is, however, phosphorus pentachloride, because the reaction in this case can be performed at low temperature increasing the stability of the intermediate formed, which presumably is an imino halide. The reaction can be performed in different solvents, but the preferred ones are chloroform and methylene chloride.

The intermediate is not isolated, but is treated with an excess of a primary alcohol to form an imino ether. The reaction temperature and the reaction time depend on the alcohol used; in most cases temperatures from -20= C. to +20 C. will be convenient.

The imino ether is not isolated, but subjected to an acid alcoholysis or hydrolysis, whereby the C N bond is cleft to yield the corresponding 6-amino-penicillanic ester of Formula VI. It is surprising that the lactam ring and the acyloxymethyl ester grouping is sufficiently stable under these conditions. By the generally used methods, the esters of 6-amino-penicillanic acid can be isolated from the reaction mixture as such or in the form of a salt with an inorganic or organic acid such as the hydrochloride or the tosylate.

The invention will now be illustrated by the following non-limiting examples:

EXAMPLE 1 (A) Acetoxymethyl ot-azido-benzylpenicillinate A mixture of potassium a-azido-benzylpenicillinate (2 g.), potassium bicarbonate (0.5 g.), acetoxymethyl bromide (1.5 ml.) and acetone (2% water) (20 ml.) was refluxed for one hour. After cooling, the suspension was filtered and the filtrate evaporated in vacuo. The oily residue was washed repeatedly by decantation with petroleum ether to yield the desired ester as a gum (2.5 g.) which did not crystallize.

(B) Acetoxymethyl a-amino-benzylpenicillinate hydrochloride To a solution of acetoxymethyl a-azido-benzylpenicillinate (2 g.) in ethyl acetate (20 ml.) was added a solution of H PO (980 mg.) and KH PO (1360 mg.) in water (20 ml.). 10% palladium on carbon catalyst (2 g.) was added, and the resulting mixture was shaken in a hydrogen atmosphere for 2 hours. The catalyst was filtered off, and the phases were separated. The aqueous phase was washed with ether, neutralized (pH 7.5) with aqueous sodium bicarbonate, and extracted several times with ethyl acetate. The combined extracts were washed with water, dried, and evaporated in vacuo. The oily residue was suspended in ethanol (10 ml.), and 2 N ethanolic hydrogen chloride (2.5 ml.) was added with stirring. Addition of ether to the resulting solution precipitated the hydrochloride of acetoxymethyl a-amino-benzylpem'cillinate, which was filtered off, washed with ether, and dried. The product thus obtained was a colourless amorphous powder, readily soluble in Water, methanol and ethanol, but sparingly soluble in ether and petroleum ether. The purity was determined iodometrically to be 86 percent. The IR. spectrum (KBr) contains strong bands at: 692 cmf 977 cmf 1020 cm.- 1195 cmr 1300 cmr 1370 cmr 1460 cmr 1500 cmr 1550 cm.- 1687 cm.- 1765 cm.-

The free base was precipitated from an aqueous solution of the hydrochloride by the addition of aqueous sodium bicarbonate, as a colourless amorphous powder. The N.M.R.-spectrum (CDCl of this product shows characteristic signals at 6:7.32 (s), 5.76 (d), 5.53, 4.52, 4.43, 2.08 (s), 1.62 (s), and 1.50 (s), TMS being used as internal reference.

EXAMPLE 2 D -u-amino-benzylpenicillin acetoxyrnethyl ester, hydro chloride D()-u-amino-benzylpenicillin (3.5 g.) and triethylamine (1.42 ml.) were mixed with acetone containing 1 percent of water (70 ml.). To the resulting solution was added potassium bicarbonate (1 g.) and bromomethyl acetate (1.75 ml.), and the mixture was stirred at room temperature for 4 hours. After filtration, the filtrate was concentrated in vacuo to about ml., ethyl acetate (100 ml.) was added, and the resulting solution was washed with aqueous sodium bicarbonate followed by water. Water ml.) was then added to the ethyl acetate solution, and, with vigorous stirring, 1 N hydrochloric acid was added drop by drop until the pH of the aqueous phase was 2.5. The aqueous layer Was separated and washed with ether. n-Butanol (150 ml.) was added, and the resulting mixture was evaporated in vacuo unt1l the water was removed. The resulting butanolic solution ml.) was poured into ether (500 ml.) whereby an amorphous precipitate separated. It was filtered off, washed with ether, and dried to yield the hydrochloride of the desired ester as a colourless product, with a purity of 83 percent (determined iodometrically). The I.R. spectrum (KBr) contains bands at: 692 cm.- 976 cm.- 1020 cm. 1195 cmr 1300 cm. 1370 ems- 1463 cm.- 1500 crnr 1550 cmr 1690 cm; 1765 cm.-

EXAMPLE 3 (A) Acetoxymethyl a-(o-nitrophenylsulphenylamino benzylpenicillinate Acetoxymethyl bromide (1.1 ml.) was added with stirring to a solution of a-(o-nitrophenylsulphenylamino)- benzylpenicillin (5.0 g.) and triethylamine (1.4 ml.) in methylene chloride ml.). The reaction mixture was stirred overnight. Thereafter it was washed with water (25 ml.), aqueous sodium bicarbonate (10 ml.), and water (10 ml.). The organic phase was dried and evaporated in vacuo to yield a gum which was washed by decantation with petroleum ether.

(B) Acetoxymethyl ot-aminobenzylpenicillinate hydrochloride To a solution of the crude acetoxyrnethyl a-(o-nitrophenyl-sulphenylamino)-benzylpenicillinate, prepared as described under A above, in ml. of ethyl acetate was added a solution of 2.2 g. of sodium thiosulphate in 50 ml. of water with vigorous stirring. The aqueous phase was separated, extracted with ether, neutralized with aqueous sodium bicarbonate (pH 7.5), and extracted twice with ethyl acetate. The organic phase was dried and evaporated in vacuo leaving an oily residue which was dissolved in ethanol (25 ml.) by the addition of 2 N alcoholic hydro: gen chloride (3 ml.) with stirring, and precipitated by addition of ether. The amorphous product was filtered oif and washed with ether, yielding the acetoxyrnethyl OL- aminobenzylpencillinate hydrochloride.

EXAMPLE 4 (A) Acetoxymethyl u-benzyloxycarbonylamino-benzylpenicillinate A mixture of potassium a-benzyloxycarbonylaminobenzylpenicillinate (2.6 g.), potassium bicarbonate (0.5 g.), acetoxyrnethyl bromide (1.5 ml.), and acetone (25 ml.) was refluxed for one hour. After cooling, the suspension was filtered and the filtrate evaporated in vacuo. The residue was dissolved in ethyl acetate and extracted with water, aqueous sodium bicarbonate, and water. The organic phase was dried and evaporated in vacuo to yield a gum which was washed by decantation with petroleum ether.

(B) Acetoxymethyl ot-aminobenzylpenicillinate hydrochloride To a solution of orthophosphoric acid (0.98 g.) and potassium dihydrogen phosphate (1.36 g.) in 25 ml. of water was added 10% palladium on barium sulphate catalyst (6 g.) and the suspension was shaken for one hour under hydrogen. A solution of the above-mentioned gum in 25 ml. of ethyl acetate was added and the resulting mixture shaken in a hydrogen atmosphere for 2 hours at room temperature and atmospheric pressure. The catalyst was filtered Off, and the aqueous phase separated and washed with ether. The aqueous phase was neutralized (to a pH about 7.5) with aqueous sodium bicarbonate and extracted twice with ethyl acetate. The combined extracts were washed with water, dried and evaporated in vacuo. Thereafter the resulting oily residue was converted to the hydrochloride of acetoxyrnethyl a-aminobenzylpenicillinate as described in Example 3B.

EXAMPLE 5 (A) Acetoxymethyl D(-)-u-azidobenzylpenicillinate Potassium D()-rx-azidobenzylpenicillinate (8.26 g.), potassium bicarbonate (1.0 g.), and bromomethyl acetate (4.1 ml.) were refluxed for one hour in a mixture of acetone (50 ml.) and water (1m1.).

After cooling, the suspension was filtered, and the filtrate evaporated in vacuo. The residue was washed repeatedly with petroleum ether to remove excess of bromomethyl acetate. The oily residue was taken up in ethyl acetate (50 ml.), and the resulting solution washed with aqueous sodium bicarbonate followed by water. After drying, the solvent was removed in vacuo to yield the desired compound as a gum.

(B) Acetoxymethyl D -a-aminobenzylpenicillinate,

- hydrochloride To a solution of acetoxyrnethyl D()-a-azidobenzylpenicillinate (10.0 g.) in ethyl acetate (150 ml.) were added water ml.) and 10% palladium on carbon catalyst (5 g.) in a flask equipped with an efiicient stirrer, a gas inlet tube, a gas outlet tube, a combined glass and calomel electrode, and a burette controlled by an auto matic titrator. The system was flushed with nitrogen whereafter a stream of hydrogen was bubbled through the suspension with stirring, a pH value of 3.0 being maintained by addition of 1 N hydrochloric acid via the automatic titrator. When the consumption of acid stopped, the flask was flushed with nitrogen until all the hydrogen was removed, and the catalyst was filtered off. The two phases of the filtrate were separated, and the aqueous phase washed with ether and freeze-dried. The desired compound was obtained as a colourless amorphous powder.

The crystalline tosylate of acetoxyrnethyl D()-a.- aminobenzylpenicillinate was obtained by adding 0.5 N aqueous sodium p-toluenesulphonate to a 20% aqueous solution of the hydrochloride. The crystalline precipitate which formed was collected, washed with water, and dried to yield colourless crystals, M.P. 166167' C.

Analysis.Calculated for C H N O S (percent): C, 52.55; H, 5.26; N, 7.09; S, 10.80. Found (percent): C, 52.80; H, 5.42; N, 6.88; S, 1064.

EXAMPLE 6 Acetoxymethyl u-amino-benzylpenicillinate To a stirred suspension of u-amino-phenylacetyl chloride hydrochloride (340 mg.) and acetoxyrnethyl 6-aminopenicillinate p-toluenesulphonate (690 mg.) in methylene chloride (8 ml.) at 0 C. was added a solution of triethylamine (0.50 ml.) in methylene chloride (2 ml.). After reaction for one hour at 0 C. and for /2 hour at room temperature, the mixture was evaporated in vacuo and treated with ethyl acetate (20 ml.) and water (25 ml.) containing 4 N hydrochloric acid (0.35 ml.). After shaking, the layers were separated, the ethyl acetate was discharged, and the pH of the aqueous solution was adjusted to 7.5 by the addition of saturated sodium bicarbonate solution (3.5 ml.). The mixture was extracted with ethyl acetate, and the organic layer was dried over magnesium sulphate and evaporated to leave the acetoxymethyl penicillin ester as a viscous oil.

The IR. spectrum (CHCl had bands at 3300 cm.- (NH), 1875 cm.- (fi-lactam), 1760 cm.- (ester carbonyl) and 1680 cm.- (amide). The identity of the compound was demonstrated by conversion of the free base into its hydrochloride and comparison with an authentic sample:

(KBr): 1765 cmf 1687 cm.- 1550 cmr 1500 cm.-

T.L.C.: R =0.51 (butanol-ethanol-H O, 8+2+2); R =0.52 (butanol-acetic acid-H O, S t-2+2) The acetoxymethyl 6-aminopenici1linate p-toluenesulphonate used as starting material was prepared in the following manner:

Acetoxymethyl 6-aminopenicillinate p-toluenesulphonate. (A) Acetoxymethyl 6-tritylaminopenicillinate A solution of 6-tritylaminopenici1lanic acid (11.5 g.) in acetone (65 ml.) was cooled to C., triethylamine (4.2 ml.), was added, followed by acetoxymethyl bromide (2.45 ml.), and the reaction mixture was kept at 0 C. with stirring for 2 hours and finally at room temperature for one hour. The triethylammonium bromide (3.5 g.), was precipitated, was removed by filtration, the evaporated filtrate was treated with ethyl acetate (175 ml.) and after 2 washings with cold 2% aqueous NaHCO and with icewater, the ethyl acetate layer was dried over MgSO, to yield, on evaporation, the desired pure acetoxymethyl ester as an amorphous powder.

Analysis-Calculated for C H N O S (percent): C, 67.77; H, 5.87; N, 5.27; S, 6.04. Found (percent): C, 67.73: H, 5.91; N, 5.22; S, 6.00.

Thm-layer chromatography (T.L.C.) on silica gel (Merck HF showed a pure product.

R =0.71 (butanol-ethanol-H o', 8+2+2).

R =0.78 (butanol-acetic acid-H O, 8 +2+2).

(B) Acetoxymethyl 6-aminopenicillinate, p-toluenesulphonate Acetoxymethyl 6-aminopenicillinate (3.71 g. in acetone (129 ml.) (0.2% H 0) was treated with anhydrous ptoluenesulphonic acid (1.21 g.). After standing for 1 /2 hours at room temperature, water (014 ml.) was added, and precipitation of the p-toluenesulphonate was brought about by addition of petroleum ether (300 ml.). Filtration and consecutive washings with acetone-petroleum ether, ethyl acetate and ether left the crude salt.

After two recrystallizations from acetone-ether, a colourless crystalline product was obtained with a melting point of: 1325- 134 C. (dec.).

Calculated for C H N O S (percent): C, 46.95; H, 5.25; N, 6.08; S, 13.92. Found (percent): C, 46.84; H, 5.17; N, 5.86; S, 13.79.

[04 +127.5 (C=1; ethanol).

The purity was further established by TLC.

R =0.51 (butanol-ethanol-H o, 8+2+2).

R =0.53 (butanol-CH OOOH-H 0, 8+2+2).

Iodometric assay showed 99% purity using 6-aminopenicillanic acid as a standard.

Acetoxymethyl-6-aminopenicillinate, p-toluenesulphonate 6-aminopenicillanic acid (4.32 g.), dispersed in acetone (140 ml.), was treated with triethylamine (6.3 ml.) with stirring at 0 C. Acetoxymethyl bromide (3.92 ml.) was added dropwise, and the mixture was kept with stirring at room temperature for 4 hours. Triethylammonium bromide (3.5 g.), which formed, was removed by filtration, and the filtrate was evaporated to dryness in vacuo, then treated with ethyl acetate (140 ml.) and cold 2% sodium bicarbonate (110 ml.). After shaking and separation, the ethyl acetate solution was further shaken with ice-water and dried over magnesium sulphate.

On evaporation, 5.2 g. of product were obtained. The crude ester, dissolved in acetone (200 ml.), was treated with anhydrous p-toluenesulphonic acid (3.5 g.) and the p-toluenesulphonate was precipitated by adding ether (750 ml.). It was collected by filtration and washed with ethyl acetate and ether to yield the desired compound.

The complete identity with the product obtained above was demonstrated by comparing melting points, IR. spectra and T.-L.C. data. IR. (KBr) showed strong bands at: 1785 cmf 1758 cmr g 1379' cmr 1325 cmr 1225 cmr 1170' cm. 1123 cur- 1030 cm. 1006 cmf 972 cmf 811 cmf 681 cmf EXAMPLE 7 Acetoxymethyl a-azido-benzylpenicillinate Acetoxymethyl 6-aminopenicillinate p-toluenesulphonate (580 mg.) was aded to a mixture of ethyl acetate ml.) and 2% aqueous sodium bicarbonate (50 ml.). a-Azido-a-phenylacetyl chloride (250 mg.) dissolved in benzene (5 ml.) was added to the mixture with vigorous stirring at 0 C. After /2 hour, the ethyl acetate layer was shaken with ice-water, dried over magnesium sulphate and evaporated in vacuo to give the desired compound. I.R. (KBr): 2125 cmf (azido group); 1780 cm.- (B-lactam); 1765 cm.- (ester carbonyl); 1690 emf (amide).

EXAMPLE 8 Acetoxymethyl a-azido-benzylpenicillinate Acetoxymethyl 6-aminopenicillinate, p-toluenesulphonate (600 mg.) was shaken with a mixture of ethyl acetate (35 ml.) and cold 2% sodium bicarbonate (17 ml.), the layers were separated, and the ethyl acetate solution was further shaken with ice-water (20 ml.), then dried over magnesium sulphate, and evaporated to leave the free acetoxymethyl ester '(360 mg.). This was dissolved in methylene chloride (8 ml.), N,N'-dicyclohexylcarbodiimide (255 mg.) was added, and the resulting solution was added quickly with stirring to a a-azido-phenylacetic acid (220 mg.) in N,N-dimethylformamide (2.5 ml.) at 0 C. The stirring was continued for one hour at room temperature, and the mixture was filtered over diatomaceous earth Dicalite (Registered trademark).

The volume of the filtrate was reduced in vacuo, ethyl acetate was added (25 ml.), and after shaking with 0.01 N hydrochloric acid and with 2% sodium bicarbonate, the ethyl acetate layer was dried over magnesium sulphate, Removal of the solvent at room temperature gave a product which proved identical with the compound obtained according to the procedure of Example 7.

EXAMPLE 9 (A) Propionyloxymethyl D(-)-a-azidobenzylpenicillinate This compound was prepared analogously to the pivaloyloxymethyl D()- x-azidobenzyl penicillinate described in Example 12A, using chloromethyl propionate as the halomethyl ester reagent.

(B) Propionyloxymethyl D(-)-a-aminobenzylpenicillinate, hydrochloride The compound was prepared by catalytic hydrogenation of propionyloxymethyl D(-)-a-azidobenzylpenicillinate in the same way as described in Example 12B.

The desired compound was obtained as a colourless, amorphous powder, and had a purity of determined iodometrically.

13 LR. (KBr): 1780 (shoulder), 1764, and 1690 cmr N.M.R. (D Signals at 6:7.94 (s.), 6.25 (d.; 1:1 c.p.s.), 5.96 (s.), 5.71 (s.), 500 (s.), 2.88 (m.; J=7 c.p.s.), 1.85 (s.), aud153 (t.; J=7 c.p.s.),TMS being used as external reference. [M 2 +191 C. (c.=1 in H O).

EXAMPLE 10 (A) Butyryloxymethyl D(')-a-azidobenzylpenicillinate The compound was prepared from -D(')-ot-8.Z1d0- benzylpenicillin potassium salt and chloromethyl butyrate in a manner analogousi with the preparation of pivaloyloxymethyl D()-ot-azidobenzylpenicillinate described in Example 12A.

(B) Butyryloxymethyl D()-otaminobenzylpenicillinate, hydrochloride The compound was prepared by catalytic hydrogenation of butyryloxymethyl D()-a-azidobenzylpenicillinate, using the method described in Example 12B, and obtained as a colourless, amorphous powder.

The purity of the compound was determined iodometrically to be 90.2%.

IR. (KBr): Bands at 1780-1775 (shoulder), 1763, and 1688 cm.- N.M. R. (D 0): Signals at 6:7.94 (s.), 6.25 (d; J=1.5 c.p.s.), 5.97 (s.), 5.73 (s.), 5.00 (s.), 2.82 (t.; J= c.p.s.), 2.04 (m.; 7.5 c.p.s.), 1.83 (s.), and 1.34 (t; I=7.5 c.p.s.), TMS was used as external reference. [a] +197 C. (c.=1 in H O).

EXAMPLE 11 (A) Isobutyryloxymethyl D 'OL'HZidObCIlZYl' penicillinate This compound was prepared from potassium D(-)- a-azidobenzylpenicillinate and chloromethyl isobutyrate in the same manner as described in Example 12A for the preparation of pivaloyloxymethyl D(-)-a-azidobenzylpenicillinate.

(B) Isobutyryloxymethyl D -a-aminobenzylpenicillinate The compound was obtained as a colourless, amorphous powder by the catalytic hydrogenation of isobutyryloxymethyl D()-ot-azidobenzylpenicillinate, using the method described in Example 12B. The compound had a purity of 92.6% determined iodometrically.

LR. (KBr): Bands at 1780 (shoulder), 1760-1755, and 1690 cmf N.M.R. (D 0): Signals at 6=7.94(s), 6.27 (d; J=1.5 c.p.s.), 5.98(s), 5.71(s), 5.01(s), 3.09 (m; J=7 c.p.s.), 1.82(s), 1.57 (d; J=7 c.p.s.), TMS was used as external reference. [u] '|196 (c.=1 in H O).

EXAMPLE 12 (A) Pivaloyloxymethyl D(- -a-azidobenzylpenicillinate To a suspension of potassium D(-)-u-azidobenzylpenicillinate(4.14 g.) and potassium dicarbonate (1.5 g.) in acetone (100 ml.) and 10% aqueous sodium iodide (2 ml.), chloromethyl pivalate (2.7 ml.) was added and the mixture refluxed for 2 hours. After cooling, the sus pension was filtered and the filtrate evaporated to dryness in vacuo. The remaining residue was washed repeatedly by decantation with petroleum ether to remove unreacted chloromethyl pivalate. The oily residue was taken up in ethyl acetate (100 ml.), and the resulting solution washed with aqueous sodium bicarbonate and water, dried and evaporated in vacuo to yield the desired compound as a yellowish gum, which crystallized from ether, M.P. 114- 115 C. [oz] Z +42 (c.=1, CHCl The LR. spectrum (KBr) contained strong bands at: 2130, 1786, 1760, 1700, 1530, 1225, 1110 and 973 cm.-

(B) Pivaloyloxymethyl D -u-aminobenzylpenicillinate, hydrochloride To a solution of pivaloyloxymethyl D(-)-u-azidobenzylpenicillinate (prepared as described above) in ethyl acetate (75 ml.) a 0.2 M phosphate buffer (pH 2.2) (75 ml.) and 10% palladium on carbon catalyst (4 g.) were added, and the mixture was shaken in a hydrogen atmosphere for 2 hours at room temperature. The catalyst was filtered off, washed with ethyl acetate (25 ml.) and phosphate buffer (25 ml.), and the phases of the filtrate were separated. The aqueous phase was washed with ether, neutralized (pH 6.57.0) with aqueous sodium bicarbonate, and extracted with ethyl acetate (2X 75 ml.). To the combined extracts, water (75 ml.) was added, and the pH adjusted to 2.5 with 1 N hydrochloric acid. The aqueous layer was separated, the organic phase extracted with water (25 ml.), and the combined extracts were washed with ether, and freeze-dried. The desired compound was obtained as a colourless, amorphous powder, soluble in Water, methanol and ethanol. The IR-spectrum (KBr) contained bands at 1780 (shoulder), 1765-1775, and 1960 CHIC-1. The NMR-spectrum (D 0) showed signals at 5=7.94(s); 6.20(rn); 5.94(s), 5.78(s), 4.96(s), 179(s), and 1.55(s), TMS being used as external reference. M1 +l (c.=1 in H O).

The purity of the compound was determined iodometrically to be 91%. A crystalline hydrochloride was obtained from isopropanol With a melting point of 155156 C. (dec.). [a] +196 (c.=1 in H O).

EXAMPLE 13 Pivaloyloxymethyl 6-aminopenicillanate, p-toluenesulphonate To a stirred solution of triethylammonium 6-tritylaminopenicillanate (5.6 g.) in acetone (25 ml.) at 0 C. was added triethylamine (1.08 ml.) followed by chloromethyl pivalate (1.6 ml.) and triethylammonium iodide (2.3 g.). Stirring was continued at 0 C. for A2 hour and at room temperature for 20 hours. The precipitate of triethylammonium chloride was removed by filtration, and the evaporated filtrate was treated with ethyl acetate (75 ml.). After shaking with cold 1% aqueous NaHCO and with ice-Water, the ethyl acetate layer was separated, dried over MgSO and evaporated in vacuo to leave the pivaloyloxymethyl ester of 6-tritylaminopenicillanic acid as an amorphous powder. To the crude ester, dissolved in ethyl acetate (40 ml.) was added a solution of p-toluenesulphonic acid, hydrate (1.90 g.) in ethyl acetate (20 ml.) at 0 C. After additional stirring for 2 hours at room temperature, the precipitate of the desired tosylate was collected on a filter and washed with ethyl acetate and ether.

Recrystallization from acetone-ether afforded the pure zrlystalline compound with a melting point of 138-39 C.

Analysis.--C H N O S requires (percent): C, 50.18; H, 6.01; N, 5.57; S, 12.76. Found (percent): C, 50.46; H, 6.15; N, 5.36; S, 12.57. [a] 1 +131 (c.=,l; ethanol).

LR. (KBr) had strong bands at: 1795, 1768, 1750, 1545, 1485, 1465, 1375, 1365, 1315, 1210, 1160, 1115, 1030, 1010, 980, 815 and 680 cmf Iodometric assay, using 6-aminopenicillanic acid as reference, showed 99% purity. The purity of the compound was further demonstrated by thin-layer chromatography (T.L.C.) on silica gel (Merck, HF

R =O.66 (n-butanol-ethanol-H O, 4+ 1 1) R =0. 12 (cyclohexane-ethyl acetate, 1+ 1 EXAMIPLE 14 Pivaloyloxymethyl 6-aminopenicillanate, p-toluenesulphonate 6-aminopenicillanic acid (4.32 g.), dispersed in acetone ml.) at 0 C., was treated with triethylamine (6.4 ml.). While maintaining stirring at 0 C., chloromethyl pivalate (5.9 ml.) and triethylammonium iodide (4.6 g) were added, and the stirred mixture was left for 20 hours at room temperature. Triethylammonium chloride (2.9 g.) was removed by filtration, and the evaporated filtrate was worked up according to the procedure described in Example 13 to yield the crude pivaloyloxymethyl ester. This was taken up in ethyl acetate (45 ml.). Treatment with anhydrous p-toluenesulphonic acid (3.4 g.) in ethyl acetate (50 ml.) caused immediate precipitation of the crystalline tosylate which was filtered off and Washed with ethyl acetate and ether, yielding a pure colourless material which in every respect proved identical with the analytically pure compound from Example 13.

EXAMPLE l Pivaloyloxymethyl 6-aminopenicillanate, p-toluenesulphonate A dispersion of 6-aminopenicillanic acid (2.16 g.) in acetone (70 ml.) at 0 C. was treated with triethylamine (3.22 ml.) followed by bromomethyl pivalate (3.92 g.) (B.P.: 50-51 C. at mm. Hg) prepared from pivaloyl bromide and paraformaldehyde by a method analogous to the preparation of chloromethyl pivalate described in J.A.C.S. 89, 5442 (1967).

After stirring for hours at room temperature, the precipitate of triethylammonium bromide (1.63 g.) was collected on a filter, and the evaporated filtrate was worked up according to the procedure described in Example 13. The crude pivaloyloxymethyl ester was dissolved in ethyl acetate (45 ml.), and crystallization of its pure tosylate was brought about by addition of anhydrous p-toluenesulphonic acid (1.72 g.) in ethyl acetate ml.). The product, which was isolated after filtration and washings with ethyl acetate and ether, showed complete identity with that of Example 13.

EXAMPLE 16 Pivaloyloxymethyl 6-aminopenicillanate, p-toluenesulphonate To 6-aminopenicillanic acid (2.16 g.) dispersed in acetone (60 ml.) at 0 C. was added triethylamine (1.8 ml.) followed by an acetone solution (10 ml.) of pivaloyloxymethyl p-toluenesulphonate (3.2 g.), The mixture was stirred for /2 hour at 0 C. and for 20 hours at room temperature. The resulting solution was evaporated in vacuo, and the residue was worked up by the method applied in Example 13. To the crude ester in ethyl acetate (75 ml.) 'was added anhydrous p-toluenesulphonic acid (1.7 g.) in ethyl acetate (25 ml.). The precipitated crystalline salt after filtration and washings with ethyl acetate proved identical with the compound prepared according to the previous Example 13.

The starting material, pivaloyloxymethyl p-toluenesulphonate, was prepared -by reacting the silver salt of ptoluenesulphonic acid with chloromethyl pivalate in dry acetonitn'le for 4 days at room temperature. It was obtained with a melting point of 4444.5 C.

Analysis.C H O S requires (percent): C, 54.53; H, 6.33; S, 11.20. Found (percent): C, 54.73; H, 6.31; S, 11.09.

EXAMPLE 17 Pivaloyloxymethyl 6-aminopenici1lanate, p-toluenesulphonate To a dispersion of 6-aminopenicillanic acid (2.16 g.) and potassium bicarbonate (1.0 g.) in acetone (70 ml.) were added chloromethyl pivalate (1.56 ml.) and potassium iodide (280 mg.). The mixture was stirred and refluxed for 5 hours. Solid material was filtered 01f, and the acetone solution was evaporated in vacuo. The residue was triturated with ethyl acetate (80 ml.), and, after filtration, the cooled ethyl acetate solution was treated with anhydrous p-toluenesulphonic acid (1.72 g.) in ethyl acetate (40 ml.). The precipitated tosylate was collected on a filter and washed with ethyl acetate and ether. Melting point, I.R. spectrum and T.L.C. data of the colourless crystalline product established the identity with the compound obtained according to previous examples.

16 EXAMPLE 1:;

Pivaloyloxymethyl 6-amin0penicillanate, hydrochloride To a solution of PCl (2.1 g.) in 20 ml. of dry, alcoholfree chloroform was added 2.26 ml. of dry quinoline with stirring. The resulting suspension was cooled to -30 C., and 4.0 g. of pivaloyloxymethyl benzylpenicillinate were added. After 30 minutes stirring at 5 C. to 10 C., the solution Was cooled to -30 C. and 6.7 ml. of dry n-propanol were added in one portion. The temperature rose to -l0 C. to 15 C. The reaction temperature was raised to 0 C. during 15 minutes and kept at this temperature for 30 minutes, wherearfter the solution was added to an ice-cold mixture of water (25 ml.) and hexane (40 ml.) with stirring. The aqueous phase was separated and the organic phase extracted three times with 25 ml. of icecold 1 N HCl. The combined aqueous phases were stirred at 0 C. with ml. of ethyl acetate, the pH being adjusted to 7.5 with NaHC'Og. The organic phase was separated, dried and evaporated in vacuo to leave an oil which was dissolved in 50 ml. of isopropanol at 0 C. By addition of 1.75 ml. of 8 N dry solution of hydrochloric acid in isopropanol with stirring, the desired hydrochloride was precipitated. It was filtered and subsequently Washed with isopropanol and ether to give a pure product with a melting point of 156-160 C. (dec.).

Analysis.Calculated for C H CIN O S (percent): C, 45.84; H, 6.32; CI, 9.66; N, 7.63; S, 8.74. Found (percent): C, 45.60; H, 6.39; Cl, 9.76; N, 7.54; S, 8.83. [a] ZI-l83 (c.=1; 0.1 N HCl). LR. (KBR) had characteristic strong carbonyl bands at 1790, 1767 and 1756 cmr- The starting material could be prepared in the followmg manner:

Pivaloyloxymethyl benzylpenicillinate To a suspension of potassium benzylpenicillinate (19.0 g.) in 200 ml. of acetone was added chloromethyl pivalate (8.3 ml.) followed by a solution of sodium iodide (1.25 g.) in water (5 ml.).. The mixture was refluxed for 5 hours. After cooling, the potassium chloride was removed by filtration. By adding water to the filtrate, the desired compound was obtained as a colourless crystalline product with a melting point of 114-1 15 C.

Analysis.Calculated for C H N O S (percent): C, 58.91; H, 6.30; N, 6.24. Found (percent): C, 58.82; H, 6.33; 'N, 6.28. [a] 2+236 (c.=1; methanol). Thinlayer chromatography on silica gel (Merck HF showed a pure product. R =0.45 (Cyclohexane-ethyl acetate, 1+1). R =0.86 (Butanol-ethanol-H O, 4+1-l-1).

In a manner analogous with that described in the procedure above, the pivaloyloxymethyl 6-aminopenicil1anate was prepared by substituting phenoxymethyl penicillin for benzylpenicillin.

EXAMPLE 19 Pivaloyloxymethyl 6-aminopenicillanate Pivaloyloxymethyl 6-aminopenicillanate, p-toluenesulphonate (24.0 g.), suspended in ethyl acetate (1.1 1.), was treated with 2% aqueous sodium bicarbonate (760 ml.) with vigorous stirring. The layers were separated, and the ethyl acetate solution was shaken thoroughly with ice-water (600 ml.) with 2% sodium bicarbonate added (25 ml.).

The ethyl acetate layer was dried over anhydrous magnesium sulphate, filtered and evaporated in vacuo. The residue was treated with petroleum ether (200' ml.) (B.P. 50 C.) and crystallization took place on stirring for 2 hours, yielding the analytically pure ester with a melting point of 60-65" C.

Analysis.C H N O S requires (percent): C, 50.90; H, 6.71; N, 8.48; S, 9.71. Found (percent): C, 51.15; H, 6.77; N, 8.36; S, 9.63. [a] 2+194- (c.=l; C H OH).

[a] 2+184 (c.=1; CHC1 LR. (KBr) showed characteristic bands at 3400, .1780, and 1750 cmr 1 7 EXAMPLE 20 Pivaloyloxymethyl D'()-a-aminobenzylpenicillanate, hydrochloride Pivaloyloxymethyl 6 aminopenicillanate, hydrochloride (7.4 g.) was suspended with eificient stirring in dry, ethanol-free chloroform (100 ml.) at C. Sodium bicarbonate (4.3 g.) was added, followed by D-()-aphenyl glycyol chloride, hydrochloride (5.0 g.), prepared according to J. Org. Chem. 31, 897 (1966).

Stirring was maintained for 3 hours at 0 C. The mix ture was filtered through diatomaceous earth, and the clear filtrate was evaporated in vacuo. The colourless residue was crystallized from isopropanol-ether, collected on a filter and washed with isopropanol and ether to yield the desired compound in high purity, showing complete identity with the hydrochloride of D()-a-aminobenzylpenicilline pivaloyloxymethyl ester which was obtained according to the procedure described in Example 12B.

EXAMPLE 21 Pivaloyloxymethyl D )-u-aminobenzylpenicillinate, hydrochloride To a solution of potassium N-[l-methyl-Z-carbethoxyvinyl]-D(-)-a-amino-a-phenylacetate (155.2 g.; 0.5 mole of the hemihydrate) in ethyl acetate (2 1.), N-methylmorpholine (2.5 ml.) and isobutyl chloroformate (70 ml.) were added at C. with stirring. Potassium chloride separated immediately, and the mixture was kept at 15 C. for 6 minutes. Then an ice-cold solution of pivaloyloxymethyl 6-aminopenicillanate in ethyl acetate (1 1.) prepared from 251.3 g. of the crystalline p-tolue'nesulphonate of this compoundwas added with stirring, the temperature being kept between -l4 C. and -12 C. during the acylation. After stirring for another 10 minutes at low temperature, the cooling bath was removed, and the mixture stirred at room temperature for 30 minutes, Thereafter the mixture was extracted with 0.5 M aqueous sodium bicarbonate (500 ml.), and washed with water (2x 250 ml.). The organic phase was dried, and the solvent removed in vacuo. The yellow only residue thus obtained was dissolved in acetone (1 1.); water (0.9 l.) was added, and 4 N hydrochloric acid was added drop by drop to the mixture with vigorous stirring. A pH-value of 2.5 was maintained in the mixture during the hydrolysis by using an automatic titrator. The reaction was finished when the consumption of hydrochloric acid ceased after addition of 100-110 ml. (80-88 percent of the theoretical amount). Acetone was removed from the mixture by evaporation in vacuo (bath-temperature about 35 C.), and the remaining aqueous phase was extracted several times with ethyl acetate. After separation of the aqueous layer, the combined ethyl acetate extracts were diluted with petroleum ether (800 ml.) and extracted with water (pH 3; 200 ml.). To the combined aqueous phases (ca. 1.21.) sodium chloride (240 g.) was added, and the mixture was shaken vigorously whereafter a yellowish organic layer was separated. The aqueous phase was further extracted with ethyl acetate (200 ml.), and the combined organic phases were dried over magnesium sulphate, filtered, whereafter isopropanol (800 ml.) was added to the filtrate. After concentration of the solution under reduced pressure to about half the volume (bath-temperature about 35 0.), another 800 ml. of isopropanol were added, and the mixture was concentrated in vacuo to about 600-800 ml. Crystalline material separated from the mixture, which was stirred for one hour at room temperature and was kept in the refrigerator overnight. The crystalline material was filtered 01f, washed with ice-cold isopropanol (100 ml.), and ether (2x 100 ml.), and dried at room temperature to yield the desired compound as colourless crystals, M.P. 155156 C. (dec.). [a] 1 +196 (c.=1 in H 0).

18 EXAMPLES 22-26 By the method of Example 15, acetoxymethyl 6-aminopenicillanate, propionyloxymethyl 6-aminopenicillanate, butyroxymethyl 6 aminopenicillanate, isobutyryloxymethyl 6-aminopenicillanate, and octanoyloxymethyl 6- aminopenicillanate were prepared by substituting the corresponding bromomethyl acylate for the bromomethyl pivalate.

EXAMPLE 27 (A) 2-ethyl-butyryloxymethyl D(-)-a-azidobenzylpenicillinate The compound was prepared from potassium D(--)-aazidobenzylpenicillinate and chloromethyl Z-ethylbutyrate in the same manner as described for the preparation of pivaloyloxymethyl D()-a-azidobenzylpenici1linate in Example 12A.

(B) Z-ethyI-butyryloxymethyl D )-a-aminobenzylpenicillinate, hydrochloride This compound was prepared by the catalytic hydrogenation of 2-ethylbutyryloxymethyl D()-a-aziodobenzylpenicillinate using the method described in Example 12B, and was obtained as a colourless amorphous powder having a purity of 86.4% determined iodometrically.

LR. (KBr): Bands at 1780 (shoulder), 1762, and 1690 cm.- N.M.R. (D 0): Signals at 6=7.92(s), 6.22(m), 5.93(s), 5.77(s), 4.96(s), 2.68(m; J=7 c.p.s.), 1.95(m; J=7 c.p.s), 1.82(s), and 1.25(t; J=7 c.p.s.), TMS was used as external reference. [u] +176 (c.=1 in H O).

EXAMPLE 28 (A) Benzoyloxymethyl D )-a-azidobenzylpenicillinate To a suspension of potassium D()-a-azidobenzylpenicillinate (4.14 g.) in a mixture of acetone ml.) and 10% aqueous sodium iodide (2 ml.), chloromethyl benzoate (2.5 g.) was added, and the mixture refluxed for 6 hours. After cooling, the suspension was filtered and the filtrate evaporated to dryness in vacuo. The residue was washed with light petroleum to remove excess of chloromethyl benzoate, and thereafter dissolved in ethyl acetate (50 ml.). The solution was washed with aqueous sodium bicarbonate followed by water, dried, and evaporated in vacuo to yield the desired compound as a gum.

(B) Benzoyloxymethyl D )-u-aminobenzylpencillinate, hydrochloride To a solution of benzolyloxymethyl D()-u-azidobenzylpenicillinate (5.0 g.) in ethyl acetate (75 ml.) was added water (50 ml.) and 10% palladium on carbon catalyst (3 g.) in a flask equipped with an eflicient stirrer, a gas inlet tube, a gas outlet tube, a glass-calomel combination electrode, and a burette controlled by an automatic titrator. The system was flushed with nitrogen whereafter a stream of hydrogen was bubbled through the suspension with stirring, a pH value of 3.0 of the aqueous phase being maintained by addition of 1 N hydrochloric acid via the automatic titrator. When the consumption of acid stopped, the flask was flushed with nitrogen until all hydrogen was removed, and the catalyst was filtered oil. The biphasic filtrate was separated and the aqueous phase washed with ether and freeze-dried. The desired compound was obtained as a colourless amorphous powder, easily soluble in water. [M 1 (c.=1, H O).

The N.M.R.-spectrum (D 0) showed signals at 6.29(m) (2 H), about 5.84(m)(3 H), 4.90(s)(1 H), 1.66(3 H) and 1.54(3 H), TMS being used as external reference.

The purity of the compound was determined iodometrically to be 92%.

19 EXAMPLE 29 (A) Z-methylbenzoyloxymethyl D(-)-a-azidobenzy1- penicillinate This compound was prepared analogous to benzoyloxymethyl D'()-ot-adizobenzylpenicillinate, using chloromethyl Z-methylbenzoate instead of chloromethyl benzoate. The chloromethyl Z-methylbenzoate was prepared from paraformaldehyde and l-methylbenzoyl chloride analogous with the preparation of chloromethyl benzoate described in J.A.C.S. 43, 662 (1921).

(B Z-methylbenzoyloxymethyl D -oz-aminobenzy1- penicillinate, hydrochloride This compound was prepared by catalytic hydrogenation of Z-methylbenzoyloxymethyl D()-a-azidobenzylpenicillinate in the same way as described in Example 283.

The desired compound was obtained as a colourless, amorphous powder, easily soluble in water. The purity of the compound was determined iodometrically to be 88%.

EXAMPLE 30 (A) 2,6-dimethylbenzoyloxymethyl D -a-azidobenzoylpenicillinate The compound was prepared from potassium D'(-)-uazidobenzylpenicillinate and chloromethyl 2,6-dimethylbenzoate as described in Example 28A. The chloromethyl 2,6-dimethylbenzoate was prepared from paraformaldehyde and 2,6-dimethylbenzoylchloride benzoate described in J.A.C.S. 43, 662 (1921).

(B) 2,6-dimethylbenzoyloxymethyl D -u-aminobenzylpenicillinate, hydrochloride This compound was prepared by catalytic hydrogenation of 2,6-dimethylbenzoyloxymeth'yl D()-u-3.Zidbenzylpenicillinate in the same way as described in Example 2813. The desired compound was obtained as a colourless, amorphous powder with a purity of 85% determined iodometrically.

EXAMPLES 3 1-5 3 Using the method described in Examples 28A and B, but substituting the XCH -OCO(CH ),,A defined in the Table IV below for the chloromethyl benzoate, the corresponding esters of D()-a-aminobenzylpen icillin are obtained.

TABLE IV 11 A Isolated as O 2,6-dlmethoxyphenyl Hydrochloride. O 2-methy1-6-ch1orophenyL Do. 0 3-trifiuoromethylphonyl Hydrobromide. O 2-nitrophenyl. Hydrochloride. O 2-methy1thiophenyl Citrate. 0 Z-thienyl Hydrobromide. O 2-furyl D0.

2 lo Do. 0 2 qulnoly Hydrochloride 1 2-thionyl Do.

1 Phonyl. Tartrate.

3 Maleate. O 3 pyrldyl Hydroiodide. 0 4-pyridyl Hydrochloride 0 Do.

0 Do. 0 Do.

1 yl Do.

1 2-methylcyclopentyL Do.

4 do D0.

0 1-b1cyclo[2.2.2]octyl Sulphate.

O l-adamanty Hydrochloride. 0 Cyclohexen-2-yl D0.

Some of the halomethyl esters used are new compounds, which as mentioned before can be prepared from paraformaldehyde and the corresponding acid halide analogous to the preparation of chloromethyl benzoate described in J.A.C.S. 43, 660 (1921).

The salts obtained according to the last column of Table IV correspond to the acids used to maintain the acidity during the hydrogenation. When e.g. hydrochloric acid is used a hydrochloride is obtained, whereas if, e.g. citric acid is used a citrate is obtained.

EXAMPLE 54 Benzoyloxymethyl D -a-aminobenzylpenicillinate, hydro chloride 'D(-)-ot-aminobenzylpenicillin (3.5 g.) and triethylamine (1.42 ml.) were mixed with acetone containing 1 percent of water (70 ml.). To the resulting solution was added potassium bicarbonate (1 g.) and bromomethyl benzoate (4.0 g.) whereafter the mixture was stirred at room temperature for 4 hours. After filtration, the filtrate was concentrated in vacuo to about 15 ml., ethyl acetate ml.) was added, and the resulting solution was washed with aqueous sodium bicarbonate followed by water. Water (30 ml.) was then added to the ethyl acetate solution, and with vigorous stirring, 1 N hydrochloric acid was added drop by drop until the pH of the aqueous phase was 2.5. The aqueous layer was separated and washed with ether; n-butanol ml.) was added, and the resulting mixture was evaporated in vacuo until the water was removed. The resulting butanolic solution (40 ml.) was poured into ether (500 ml.) whereby an amorphous precipitate separated. It was filtered oif, washed with ether and dried to yield the hydrochloride of the desired ester as a colourless product, identical with the compound of Example 28B.

By the methods of Examples 19, 20 and 54 the compounds of Table N can also be obtained.

EXAMPLE 55 Mixture containing pivaloyloxymethyl D()-a-aminobenzylpencillinate, hydrochloride Aluminium monostearate (75 g.) was dissolved in modified coconut oil (4300 g.) by heating to 140 C. After cooling, flavour (15 g.) was added. Saccharose (500 g.) and pivaloyloxymethyl D()-a-aminobenzylpenicillinate hydrochloride g.) were mixed, reduced by milling to a particle size of about 10 micron and mixed with the oily solutionby means of a high speed stirrer.

A mixture was obtained containing 35 mg. of pivaloyloxymethyl D()-ot-aminobenzylpenicillinate hydrochloride per ml.

We claim:

1. A pharmaceutical composition in dosage unit form for the treatment of patients suifering from infectious bacterial diseases, which comprises as a therapeutically active ingredient, a compound selected from the group consisting of the individual isomers, mixtures of isomers of esters of a-aminobenzylpenicillin of the formula:

in which the asterisk denotes an asymmetric carbon atom and A is an alkyl radical having 1 to 6 carbon atoms, and salts of these esters with pharmaceutically acceptable acids, the compound being admixed with an atoxic, phar= 22 maceutically acceptable carrier, and the dosage unit con- References Cited T 1 d 1 h A 3,120,514 2/1964 Doyle", et al. 260-4391 i f as mm m c 1 W 5 3,228,930 1/1966 Sjobeifg et a1. 260239.1 3. A composition as claimed in claim 1 wherein the 3,262,928 7/1966 i f dosage unit 1s m the form of a tablet. ALBERT T. MEYERS m y Examiner 4. A composition as claimed in claim 1 wherein the dosage unit is in the form of a capsule. D. M. STEPHENS, A sta Examiner 

